Mammalian Cell Lines for Increasing Longevity and Protein Yield from a Cell Culture

ABSTRACT

Disclosed are compositions and methods for increasing the longevity of a cell culture and permitting the increased production of proteins, preferably recombinant proteins, such as antibodies, peptides, enzymes, growth factors, interleukins, interferons, hormones, and vaccines. Cells transfected with an apoptosis-inhibiting gene or vector, such as a triple mutant Bcl-2 gene, can survive longer in culture, resulting in extension of the state and yield of protein biosynthesis. Such transfected cells exhibit maximal cell densities that equal or exceed the maximal density achieved by the parent cell lines. Transfected cells can also be pre-adapted for growth in serum-free medium, greatly decreasing the time required to obtain protein production in serum-free medium. In certain methods, the pre-adapted cells can be used for protein production following transformation under serum-free conditions. The method preferably involves eukaryotic cells, more preferably mammalian cells.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/287,395, filed Nov. 2, 2011, which is a divisional of U.S. patentapplication Ser. No. 12/819,533 (now issued U.S. Pat. No. 8,076,140),filed Jun. 21, 2010, which is a divisional of U.S. patent applicationSer. No. 12/405,733 (now issued U.S. Pat. No. 7,785,880), filed Mar. 17,2009, which is a divisional of U.S. patent application Ser. No.11/487,215 (now issued U.S. Pat. No. 7,537,930), filed Jul. 14, 2006,which is a continuation-in-part of U.S. patent application Ser. No.11/187,863 (now issued U.S. Pat. No. 7,531,327), filed Jul. 25, 2005,which claimed the benefit under 35 U.S.C. §119(e) of provisional U.S.Patent Application No. 60/590,349, filed Jul. 23, 2004, each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Various embodiments of the present invention concern methods andcompositions for increasing longevity and/or protein yield from a cellline. In particular embodiments, the cell line may be a hybridoma cellline that produces antibodies or antibody fragments. In more particularembodiments, the methods may comprise transfecting a cell line with oneor more genes, such as genes encoding E6, E7 and/or Bcl-2 or relatedproteins. Such proteins are not limited to their native sequence, butmay include one or more substituted amino acids, such as a Bcl-2 withpoint mutations at T69E, S70E and S87E. Other embodiments concernmammalian cell lines that are capable of growth and protein productionin serum-free medium. Such cell lines may be used in methods of proteinproduction, by transfecting the cell line with an expression vector thatexpresses a heterologous protein, such as an antibody, bispecificantibody, multivalent antibody or multispecific antibody or fragmentthereof. In preferred embodiments, the cell line may be transfected inserum-free medium, providing considerable time savings in avoidinghaving to adapt the transfected cell line for serum-free growth andprotein production.

BACKGROUND OF THE INVENTION

Culturing cells in vitro, especially in large bioreactors, has been thebasis of the production of numerous biotechnology products, and involvesthe elaboration by these cells of protein products into the supportmedium, from which these products are isolated and further processedprior to use clinically. The quantity of protein production over timefrom the cells growing in culture depends on a number of factors, suchas, for example, cell density, cell cycle phase, cellular biosynthesisrates of the proteins, condition of the medium used to support cellviability and growth, and the longevity of the cells in culture (i.e.,how long before they succumb to programmed cell death, or apoptosis).Various methods of improving the viability and lifespan of the cells inculture have been developed, together with methods of increasingproductivity of a desired protein by, for example, controllingnutrients, cell density, oxygen and carbon dioxide content, lactatedehydrogenase, pH, osmolarity, catabolites, etc. For example, increasingcell density can make the process more productive, but can also reducethe lifespan of the cells in culture. Therefore, it may be desirous toreduce the rate of proliferation of such cells in culture when themaximal density is achieved, so as to maintain the cell population inits most productive state as long as possible. This results inincreasing or extending the bioreactor cycle at its production peak,elaborating the desired protein products for a longer period, and thisresults in a higher yield from the bioreactor cycle.

Many different approaches have been pursued to increase the bioreactorcycle time, such as adjusting the medium supporting cell proliferation,addition of certain growth-promoting factors, as well as inhibiting cellproliferation without affecting protein synthesis. One particularapproach aims to increase the lifespan of cultured cells via controllingthe cell cycle by use of genes or antisense oligonucleotides to affectcell cycle targets, whereby a cell is induced into a pseudo-senescencestage by transfecting, transforming, or infecting with a vector thatprevents cell cycle progression and induces a so-called pseudo-senescentstate that blocks further cell division and expands the proteinsynthesis capacity of the cells in culture; in other words, thepseudo-senescent state can be induced by transfecting the cells with avector expressing a cell cycle inhibitor (Bucciarelli et al., U.S.Patent Appl. 2002/0160450 A1; WO 02/16590 A2). The latter method, byinhibiting cell duplication, seeks to force cells into a state that mayhave prolonged cell culture lifetimes, as described by Goldstein andSingal (Exp Cell Res 88, 359-64, 1974; Brenner et al., Oncogene17:199-205, 1998), and may be resistant to apoptosis (Chang et al., ProcNatl Acad Sci USA 97, 4291-6, 2000; Javeland et al., Oncogene 19, 61-8,2000).

Still another approach involves establishing primary, diploid humancells or their derivatives with unlimited proliferation followingtransfection with the adenovirus E1 genes. The new cell lines, one ofwhich is PER.C6 (ECACC deposit number 96022940), which expressesfunctional Ad5 E1A and E1B gene products, can produce recombinantadenoviruses, as well as other viruses (e.g., influenza, herpes simplex,rotavirus, measles) designed for gene therapy and vaccines, as well asfor the production of recombinant therapeutic proteins, such as humangrowth factors and human antibodies (Vogels et al., WO 02/40665 A2).

Other approaches have focused on the use of caspase inhibitors forpreventing or delaying apoptosis in cells. See, for example, U.S. Pat.No. 6,586,206. Still other approaches have tried to use apoptosisinhibitors such as members of the Bcl-2 family for preventing ordelaying apoptosis in cells. See Arden et al., Bioprocessing Journal,3:23-28 (2004). These approaches have yielded unpredictable results. Forexample, in one study, expression of Bcl-2 increased cell viability butdid not increase protein production. (See Tey et al., Biotechnol.Bioeng. 68:31-43, 2000.) Another example disclosed overexpression ofBcl-2 proteins to delay apoptosis in CHO cells, but Bcl-xL increasedprotein production whereas Bcl-2 decreased protein production (seeWO03/083093). A further example disclosed experiments using expressionof Bcl-2 proteins to prolong the survival of Sp2/0-Ag14 (ATCC #CRL-1581, hereafter referred to as Sp2/0) cells in cultures. However,the cell density of the Bcl-2 expressing clones were 20 to 50% lowerthan that of their parental cultures, raising concerns for theirpractical application in biopharmaceutical industry (see WO03/040374;U.S. Pat. No. 6,964,199).

It is apparent, therefore, that improved host cells for high levelexpression of recombinant proteins and methods for reliably increasingrecombinant protein production, in particular the production ofantibodies and antibody fragments, multispecific antibodies, fragmentsand single-chain constructs, peptides, enzymes, growth factors,hormones, interleukins, interferons, and vaccines, in host cells areneeded in the art. A need also exists for cell lines that arepre-adapted to grow in serum-free or serum-depleted medium, that can betransfected with expression vectors under serum free conditions and usedfor protein production without going through a lengthy adaptation periodbefore serum-free growth and protein production.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideimproved host cells and methods to increase the longevity and/orrecombinant protein yields of a cell culture by introducing into thecells agents that inhibit senescence or that promote cell survival,e.g., anti-apoptotic agents. The use of such agents preferentiallyincreases the lifespan and viability of cells in culture used for theproduction of a desired recombinant protein, concomitantly increasingthe productivity of such cells in culture, and thereby the optimal yieldof the desired protein. Preferably, the apoptosis inhibitors used in themethod of the present invention include but are not limited to Bcl-2 andits family members. Alternately, the longevity and recombinant proteinyields of a cell clone can be improved by introducing into the cellagents that down-regulate the level of intracellular pro-apoptoticproteins, such as p53 and Rb, or up-regulate intracellularanti-apoptotic proteins, such as Bcl-2.

Preferably, the regulatory agents used in the method of the presentinvention include, but are not limited to, human papillomavirus type 16(HPV-16) oncoproteins E6 and E7, anti-apoptosis protein Bcl-2 andcombinations thereof. Additionally, caspase inhibitors, as describedherein, may also contribute to blocking or reducing apoptosis, thusincreasing cell survival and increasing the production of recombinantproteins by said cells in culture. A further class of anti-apoptoticagents that can be used in these cultures to enhance production ofrecombinant proteins includes certain members of the cytokine type Isuperfamily, such as erythropoietin (EPO). EPO, as a prototype moleculeof this class, is a major modifier of apoptosis of multiple cell types,not just erythrocytes, and thus has more general cytoprotectivefunctions, such as in endothelial cells, myocardial cells, tubularepithelial cells of the kidney, skin, and neurons [cf. review by P.Ghezzi and M. Brines, Cell Death and Differentiation 11 (suppl. 1),s37-s44, July 2004].

In various embodiments, the cell lines that have been transfected withone or more regulatory agents, such as HPV-16, E6, E7 and/or Bcl-2 maybe pre-adapted for growth in serum-free medium. Such pre-adapted celllines, including but not limited to the Sp/ESF cell line (see Examplesbelow), are able to undergo further transformation, under serum-freeconditions, with one or more expression vectors, thus allowingexpression and protein production under serum-free conditions withoutextensive time required for adaption to serum-free growth. Thissurprising result allows protein production under serum free or lowserum conditions, providing significant savings on medium cost. At thesame time, transfection and protein production under serum-freeconditions saves substantial time needed for serum-free adaptation thatis required when using standard mammalian cell lines, which are onlytransfectable under serum-rich conditions and require an additional 6 to12 months to adapt to serum-free protein production.

The present invention also teaches cell culture methods incorporatingnovel combinations of factors including, but not limited to,transfection vectors, screening and selection of cell clones withdesired properties, cell culture media, growth conditions, bioreactorconfigurations, and cell types to create cell culture conditions inwhich the longevity of the cell culture is increased and/or made optimaland the yield of a desired recombinant protein is increased. These cellculture methods include suspension, perfusion, and fed-batch methods ofproduction. See Tey et al., J. Biotechnol. 79: 147-159 (2000); Zhang etal., J. Chem. Technol. Biotechnol. 79: 171-181 (2004); Zhou et al.,Biotechnol. Bioeng. 55: 783-792 (1997).

Unless otherwise defined, all technical and scientific terms used hereinhave their plain and ordinary meaning. In addition, the contents of allpatents and other references cited herein are incorporated by referencein their entirety.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

FIG. 1 shows visual images of Sp2/0 and Sp-E26 cells treated withcycloheximide (+CHX) or untreated (−CHX).

FIG. 2 shows the results of screening HPV E6/E7 transduced cells thatare more resistant to CHX treatment. A total of 55 clones were screened;in the first experiment, 31 clones were screened (top panel); in thesecond experiment, 24 clones were screened (bottom panel). Healthy cellsof each clone were split into two equal portions. One was treated withCHX for 2 h and the other left untreated. The viable cells in these twocultures were then measured by MTT assay and the ratios of viable cellpopulations treated (CHX.sup.+) vs. untreated (CHX.sup.−) were plotted.As shown in the top panel, CHX treatment resulted in 30% reduction ofviability in Sp2/0 cells, while only 6% reduction in Sp-E26 cells. Sevenof the 31 clones screened (indicated by *) performed significantlybetter (<20% reduction of viability) than Sp2/0 but not as well asSp-E26. For the 24 clones screened in the second experiment (bottompanel), CHX treatment resulted in about 50% reduction of viability inSp2/0 cells and <20% reduction of viability in Sp-E26. Ten of the 24clones (indicated by * or **) screened performed significantly better(<30% reduction) than Sp2/0, and 6 of them (indicated by **) matched orwere better than Sp-E26 (<20%). E28 and E36 are two additional controlclones that perform better than Sp2/0 but not as well as Sp-E26.

FIG. 3 shows the dot plots of Guava Nexin V assay. The percentage ofearly apoptotic cells (Nexin V-positive and 7-AAD-negative) is indicatedin the lower-right quadrant.

FIG. 4 shows the DNA fragmentation in Sp2/0 treated by CHX. In contrast,Sp-E26 cells are resistant to the treatment.

FIG. 5 shows the growth profiles of Sp2/0 and Sp-E26 cells in T-flasks.Healthy cells (>95% viability) were seeded in T-flasks at an initialcell density of 200,000/ml. Viable and dead cells were counted dailyusing Guava ViaCount reagent (Guava technologies, Inc.) and PCAinstrumentation (Guava Technologies, Inc.). Accumulation of NH₄ ⁺ andlactate also was monitored.

FIG. 6 compares the growth profiles of Sp2/0 and Sp-E26 cells asdetermined for a batch culture in 3-L bioreactors. Healthy cells (>95%viability) were seeded in the bioreactors at an initial cell density of250,000/ml. Cells were counted daily by trypan blue and microscope.

FIG. 7 shows a representative immunoblot stained with Bcl-2 (100)antibody (Santa Cruz Biotech.) and developed with enhancedchemiluminescence for screening of clones for Bcl-2-EEE expression.

FIG. 8 shows a graph of flow cytometry results using Guava Express.Cells were fixed and permeabilized before staining withphycoerythrin-conjugated anti-Bcl-2 antibody (Santa Cruz Biotechnology,Inc.) Several sub-clones are compared.

FIG. 9 shows a graph of flow cytometry results using Guava Express.Cells were fixed and permeabilized before staining with phycoerythrinconjugated anti-Bcl-2 antibody (Santa Cruz Biotechnology, Inc.). Sp2/0,Raji and Daudi cells were compared to Bcl-2-EEE clones.

FIG. 10 shows the results of immunoblot analyses of 665.B4.1C1, Sp2/0,Raji, Daudi, Sp-EEE (87-29 clone) and Sp-EEE (7-16 clone) cell lysates.(A) Blots stained with a human Bcl-2 specific antibody (Santa CruzBiotechnology, Inc). (B) Blot stained with an anti-Bcl-2 antibody (SantaCruz Biotechnology, Inc) that recognizes mouse and human Bcl-2.

FIG. 11 shows growth curves (A) and viability (B) of Sp-EEE clonescompared to Sp2/0 cells grown in media supplemented with 10% fetalbovine serum.]

FIG. 12 shows growth curves (A) and viability (B) of Sp-EEE clonescompared to Sp2/0 cells grown in media supplemented with 1% fetal bovineserum.

FIG. 13 shows growth curves (A) and viability (B) of Sp-EEE clonescompared to Sp2/0 cells grown in serum-free media.

FIG. 14 shows methotrexate kill curves for Sp-EEE (87-29 clone) cells.

FIG. 15 shows a graph of flow cytometry results using Guava Expresscomparing Sp-EEE clones grown in the presence or absence of 1 mg/mlzeocin. Cells were fixed and permeabilized before staining withphycoerythrin conjugated anti-Bcl-2 antibody (Santa Cruz Biotechnology,Inc).

FIG. 16 shows the map of the pdHL2 vector used to transfect Sp2/0 cellsto obtain the 665.2B9 clone with humanized antibody sequences and theSV40 promoter and enhancer sequences.

FIG. 17 shows the map of DNA plasmid with incorporated Bcl-2 gene, usedfor transfection of clone 665.2B9

FIG. 18 and FIG. 19 show the growth profiles of Bcl-2 transfected clones665.2B9#4, Bcl-2 negative clones and untransfected control. Healthycells (>95% viability) were seeded in 24-well plates at an initial celldensity of 400,000/ml. Viable and dead cells were counted daily usingGuava ViaCount reagent and PCA instrumentation.

FIG. 20 and FIG. 21 show growth profiles of Bcl-2 transfected clone665.259#4 and Bcl-2 negative clones in different MTX concentration.Healthy cells (>95% viability) were seeded in T-flasks at initial celldensity of 100,000/ml. Viable cell density and viability were counteddaily using Guava ViaCount reagent and PCA instrumentation.

FIG. 22 shows the levels of human Bcl-2 expressed by clone 665.2B9#4 inincreasing concentrations of MTX and clone #13 detected by Westernblotting.

FIG. 23 and FIG. 24 show the profiles of cell viability and viable celldensity, respectively, of clone 665.2B9#4 cultured in 0.6 and 1.mu.M ofMTX and the Bcl-2-negative clone #13 cultured in 0.3.mu.M MTX with orwithout spiking L-glutamine and glucose. Healthy cells (>95% viability)were seeded in roller bottles at an initial cell density of 200,000/ml.On day 2 and 4 (arrows indicated), a nutrient supplement solutioncontaining glucose and L-glutamine was added to the “spiked” culture.Viable and dead cells were counted daily using Guava ViaCount reagentand PCA instrumentation.

FIG. 25 shows the viability in serum-free medium of adapted Sp/EEEsub-clones over 5 days of culture.

FIG. 26 illustrates the viable cell density of serum-free Sp/EEEsub-clones over 5 days of culture.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

As used herein, the term “about” means plus or minus ten percent. I.e.,“about 100” means a number between 90 and 110.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion or analog of an immunoglobulin molecule, like an antibodyfragment.

An antibody fragment is a portion of an antibody such as F(ab)₂,F(ab′)₂, Fab, Fv, sFv and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains, recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”), and minimal recognition units (CDR) consisting of theamino acid residues that mimic the hypervariable region.

As used herein, the term antibody fusion protein refers to arecombinantly produced antigen-binding molecule in which two or more ofthe same or different scFv or antibody fragments with the same ordifferent specificities are linked. Valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindsto one such epitope, for example a diabody with two binding sitereactive with the same antigen. The fusion protein may comprise a singleantibody component, a multivalent or multispecific combination ofdifferent antibody components, or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise an antibody oran antibody fragment and a therapeutic agent. Examples of therapeuticagents suitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

Cell Lines

Various embodiments of the present invention concern improvedcompositions, including host cell lines, and methods for enhancedproduction of recombinant proteins in such cell lines. Cell lines havebeen created that constitutively express one or more anti-apoptoticgenes and that can be transfected with an expression construct encodinga protein or peptide of interest, where expression of the anti-apoptoticgene(s) prolongs survival of the transfected cell in culture andprovides for enhanced yields of the protein or peptide of interest.

Specific embodiments concern derivatives of the Sp2/0 myeloma cell linethat provide novel cell lines, referred to as Sp-E26, Sp-EEE and Sp-ESF,which show enhanced survival in batch culture. Sp-E26 constitutivelyexpresses the E6 and E7 proteins of HPV-16. Sp-EEE and Sp-ESFconstitutively express a Bcl-2 mutant, referred to as Bcl-2-EEE. Inaddition, recombinant protein production, and particularly production ofrecombinant antibodies and antibody fragments, can be improved upontransfecting Sp-E26, Sp-EEE or Sp-ESF with an expression vector for therecombinant protein of interest. The E6/E7 or Bcl-2-EEE proteins delayinduction of apoptosis in the host cells and permit enhanced recombinantprotein production in the host cells. Protein production can be boostedstill further by addition of one or more caspase inhibitors (e.g.,caspase 1 and/or 3 inhibitors) (Bin Yang et al. Nephron ExperimentalNephrology 2004; 96:e39-e51), and/or by addition of one or more membersof the cytokine type I superfamily, such as erythropoietin (EPO), intothe growth medium of the cells. A pan-caspase inhibitor is particularlyeffective in this regard.

Further, the Sp-EEE cell line can be pre-adapted for growth and proteinproduction in serum-free or low-serum conditions, resulting inserum-free pre-adapted cell lines such as Sp-ESF. The Sp-ESF and similarcell lines may be transfected with one or more expression vectorsencoding a protein of interest, such as an antibody, antibody fragment,bispecific antibody, etc. The use of serum-free conditions fortransfection, which is unique among mammalian cell lines available fortransfection and protein production, saves a significant amount of timerequired for adaptation to serum-free growth.

Production of recombinant proteins, such as antibodies or antibodyfragments, can be significantly enhanced in the host cell byco-expression of an apoptosis inhibitor, such as Bcl-2. In particular,protein production is significantly enhanced in a myeloma cell line,such as Sp2/0, that is stably transfected with an expression vectorencoding an antibody or antibody fragment and that is co-transfectedwith an expression vector encoding an apoptosis inhibitor, such asBcl-2. Increased production of antibody can also be obtained from a hostcell transfected with the E6/E7 gene. Recombinant protein production canbe boosted still further by addition of one or more caspase inhibitorsinto the growth medium of the cells. A pan-caspase inhibitor isparticularly effective in this regard. Also, recombinant proteinproduction can be enhanced by feeding EPO, or another anti-apoptoticcytokine, into the medium of the cell culture.

Physiological, or programmed, cell death, referred to as apoptosis (Kerret al., Br J. Cancer., 26:239-257, 1972) is essential for proper tissuedevelopment and maintenance and is controlled by an intrinsic geneticprogram that has been conserved in evolution (Ellis et al., Annu RevCell Biol, 7, 663-698, 1991). Hence, when cells grow in artificialenvironments, such as ex vivo cultures, this genetic endowment resultsin a finite lifespan. Therefore, the utility of such cell cultures forthe production of proteins used in medicine and industry, as well asresearch, is dependent on maintaining such cultures for extendedlifespan, or cycles, before they die according to apoptotic mechanisms.

Methods and agents have been discovered that act independently on cellproliferation and cell death events, by differentiating cell cycle fromapoptotic effects. Bcl-2, a well-known intracellular regulator ofapoptosis (Vaux et al., Nature 335, 440-2, 1988), is a proto-oncogenethat has been found to have an anti-apototic effect that is geneticallydifferent from its inhibitory influence on cell cycle entry (Huang etal., EMBO J 16, 4628-38, 1997). Two homologues of Bcl-2, Bcl-x_(L) andBcl-w, also extend cell survival, but other members of the Bcl-2 family,such as Bax and Bak, are pro-apoptotic (Oltvai et al., Cell 74, 609-19,1993; Chittenden et al., Nature 374, 733-6, 1995; Farrow et al., Nature374, 731-3, 1995; Kiefer et al., Nature 374, 736-9, 1995). Otheranti-apoptotic genes include Bcl-6 and Mcl-1.

Thus, Bcl-2 and certain of its family members exert protection againstapoptosis, and it may be used in a method to increase the lifespan ofcertain host cells in culture that are used for the production ofproteins, thereby enhancing the amount of proteins produced andisolated. Over-expression of an anti-apoptotic Bcl-2 family member, suchas Bcl-2, BCl-x_(L), Bcl-w or mutant varieties of these proteins,inhibits apoptosis, resulting in increased cell density and longerculture survival. Hence, transfection of anti-apoptotic Bcl-2 familygenes avoids the necessity to prolong the cell culture by interferingwith the cell cycle per se, as others have proposed (ibid.). Similarly,transfection of fibroblasts with genes for Bcl-2 results inover-expression of Bcl-2 in these cells, resulting in an antagonism ofapoptosis and increasing the lifespan of these cells, with a concomitantincrease in the production and isolation of recombinant proteins. It hasalso been observed that upon cytokine withdrawal, interleukin-6(IL-6)-dependent murine myeloma cells expire as if they undergoapoptosis. It was also found that IL-6-receptors in such cells could beregulated by Bcl-2 or Bcl-x_(L) in extending apoptosis (Schwarz et al.,Cancer Res 55:2262-5, 1995).

It has been reported that a mutant Bcl-2 possessing three pointmutations (T69E, 570E and S87E) exhibited significantly moreanti-apoptotic activity compared to wild type or single point mutants(Deng et al., PNAS (101) 153-158, 2004). Thus, various embodimentsconcern the construction of an expression vector for a Bcl-2-EEE triplemutant, which was then used to transfect Sp2/0 cells to create Sp-EEEclones and subclones that show improved longevity and recombinantprotein production.

Other agents, such as oncogenic viruses, can also oppose apoptosis aspart of their eliciting cellular immortalization and ultimately completemalignant transformation, such as high-risk type HPV oncoproteins E6 andE7 (Finzer et al., Cancer Lett 188, 15-24, 2002). For example, the viralE6 protein effectively blocks the epidermal apoptotic response toultraviolet light (Storey, Trends Mol Med 8, 417-21, 2002). It has alsobeen suggested, from indirect evidence, that the human papillomavirusmay cause reduced apoptosis in squamous (but not basal cell) carcinoma(Jackson et al., Br J Cancer 87, 319-23, 2002). However, not allpapillomavirus oncoproteins have anti-apoptotic effect. For example,other studies have reported that the papillomavirus E6 protein of bovinespecies sensitizes cells to apoptosis (Liu et al., Virology 295, 230-7,2002), which is in contrast to other studies showing that HPV-16 E7 geneprotects astrocytes against apoptosis induced by certain stimuli (Lee etal., Yonsei Med J 42, 471-9, 2001). By use of E6-binding peptideaptamers, direct experimental evidence was obtained that HPV E6oncoprotein has anti-apoptotic activity in HPV-positive tumor cells(Butz et al., Proc Natl Acad Sci USA 97, 6693-7, 2000). However, otherHPV oncoproteins can have the opposite effect. The E2 protein inducesapoptosis in the absence of other HPV proteins (Webster et al., J BiolChem 275, 87-94, 2000).

Continuous expression of both the E6 and E7 proteins is known to berequired for optimal proliferation of cervical cancer cells and the twoviral proteins exert distinct effects on cell survival (DeFilippis etal., J Virol 77, 1551-63, 2003). The primary intracellular targetattributed to HPV-16 E6 is p53. E6 forms a ternary complex with p53 anda cellular ubiquitin ligase, E6AP, resulting in the ubiquitination anddegradation of p53 through the proteosome pathway and inactivation ofp53. On the other hand, HPV-16 E7 protein interacts and destabilizes thetumor suppressor protein Rb. Moreover, levels of a variety of otherintracellular proteins involved in apoptosis and cell cycle pathwayswere reported to be regulated by E6 and E7 transformation, such asBcl-2, Bcl-x_(L), p73, MDM2, p21, cyclins and cdc, cdk proteins, etc.Changes in the expression of these proteins will greatly influence thephysiological properties of the cell. The present inventors thereforehypothesized that transfection of cells in culture by HPV-16E6 and E7would be effective in generating genetically modified clones that areresistant to aging-culture-condition induced apoptosis and, therefore,prolong the lifespan of the cell culture. It was also postulated thatintroduction into a cell of either HPV-16 oncoprotein E7 or E6 alonemight be sufficient to generate genetically modified clones withimproved resistance to aging-culture-condition induced apoptosis. Whenthe cell is a recombinant protein-producing clone, the improvedphysiological properties would in turn translate into enhanced overallprotein productivity.

Generation of New Host Cells Expressing Viral Anti-Apoptotic Genes

Host cells, such as myeloma host cells, can be generated thatconstitutively express viral anti-apoptotic genes, such as HPV-16 E6 andE7 proteins. These host cells can be transfected with an expressionvector that encodes a recombinant protein of interest and co-expressionof the anti-apoptotic genes results in significantly increasedproduction of the recombinant protein.

The host cell can be essentially any host cell suitable for recombinantprotein production that can be stably transformed with the viralanti-apoptosis genes. For many recombinant proteins, host cells such asCHO and COS cells are advantageous, while for other proteins, such asantibodies, host cells such as myeloma cells and CHO cells are thecommon choices. Other examples of useful host cell lines are VERO andHeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7,293, HepG2, 3T3, NSO, NS1, RIN and MDCK cell lines. Cell lines of usemay be obtained from commercial sources, such as the COS-1 (e.g., ATCCCRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCCCRL-10), P3X3Ag8.653 (ATCC CRL-1580), CHO (e.g., ATCC CRL 1610) andBSC-1 (e.g., ATCC CRL-26) cell lines. The viral (e.g., E6/E78) and/oreukaryotic genes can be introduced into the host cell by any suitablemethod that results in constitutive or inducible expression of thegenes, i.e., any method that permits stable integration of the genesinto the host cell chromosome while permitting expression of the genes.Methods for stable transformation of host cells with a gene of interestare well known in the art. A particularly advantageous method is to usea retroviral vector that encodes the viral anti-apoptosis genes.Suitable vectors include the LSXN vector (Miller et al. Biotechniques 7,980-90, 1989). However, any alternative methods known in the art, suchas electroporation or cell fusion, may be utilized.

Advantageously, the vector used to transfect the host cell contains aselectable marker that permits selection of cells containing the vector.Suitable selection markers, such as enzymes that confer antibioticresistance on transfected cells, are well known in the art. Aftertransfection, cells are maintained in a medium containing the selectionagent, such as an antibiotic, and screened for resistance to the marker.Cells can be selected and cloned by limiting dilution using conventionalmethods.

The ability of the viral anti-apoptosis genes to increase cell viabilitycan be tested by challenging the cells with an agent that inducesapoptosis, such as cycloheximide (CHX). Cells that do not express theviral anti-apoptosis genes tend to demonstrate significant onset ofapoptosis, whereas cells expressing the genes exhibit drasticallyreduced apoptotic activity. Methods of detecting apoptosis are wellknown in the art and include, for example, cell surface FITC-Annexin Vbinding assay, DNA laddering assay and TUNEL assay.

Upon selection of suitable cells expressing the viral anti-apoptosisgenes, the cells can be transfected with an expression vector encodingthe recombinant protein of choice. The expression vector can be a vectorsuitable for transient expression or, advantageously, can be an episomalvector containing a eukaryotic origin of replication, or an amplifiablevector that permits stable integration and subsequent gene amplificationof the expression cassette. Suitable vectors are well known in the artand include, for example, the pdHL2 vector, which is particularly suitedfor production of antibodies and antibody fragments. When an amplifiableexpression cassette is used, it advantageously contains a selectablemarker that is different from the selectable marker used in theretroviral vector, to allow selection of transfected cells. Once again,suitably transfected cells can be selected and then cloned by limitingdilution.

Upon selection of suitable clones, the cells can be placed in a suitablemedium and cultured to produce the desired protein of interest. Themedium can contain serum or, preferably, be serum-free. In addition,cell longevity and protein production also can be increased by addingone or more caspase inhibitors (e.g., caspase 1 or 3) to the culturemedium. Preferably the caspase inhibitor acts to inhibit one or more ofcaspase 3, caspase 9 and/or caspase 12. A cell-penetrating caspaseinhibitor advantageously is used, and a pan-caspase inhibitor isparticularly advantageous. Suitable inhibitors such as Z-VAD-fmk andAc-DEVD-cho (SEQ ID NO: 7) are well known in the art. Alternatively, thecell line can be further transfected to express a caspase inhibitor,such as Aven or XIAP, to enhance its growth properties by affectingapoptosis. In this regard, certain members of the cytokine type Isuperfamily, such as EPO, can also increase cell survival by havinganti-apoptotic and cytoprotective actions.

The methods described above generate a cell line that can be used fortransfection with essentially any desired gene. However, the skilledartisan will recognize that established cell lines that constitutivelyexpress a desired protein, and particularly a recombinant protein, canbe subsequently transfected with a suitable vector encoding the viral orBcl-2 family anti-apoptosis genes. See Example 2 below.

The protein of interest can be essentially any protein that can beproduced in detectable quantities in the host cell. Examples includetraditional IgG type antibodies, Fab′, Fab, F(ab′)₂ or F(ab)₂ fragments,scFv, diabody, IgG-scFv or Fab-scFv fusion antibodies, IgG- orFab-peptide toxin fusion proteins, or vaccines [e.g., including notlimited to, Hepatitis A, B or C; HIV, influenza viruses, respiratorysyncytial virus, papilloma viruses, Herpes viruses, Hantaan virus, Ebolaviruses, Rota virus, Cytomegalovirus, Leishmania RNA viruses, SARS,malaria, tuberculosis (Mycobacteria), Anthrax, Smallpox, Tularemia, andothers listed in www.vaccines.org, incorporated herein by reference inits entirety]. The host cells described herein are particularly suitablefor highly efficient production of antibodies and antibody fragments inmyeloma cell lines as described in Examples 1 and 2, as well asrecombinant growth factors (e.g., EPO, G-CSF, GM-CSF, EGF, VEGF,thrombopoietin), hormones, interleukins (e.g., IL-1 through IL-31),interferons (e.g., alpha, beta, gamma, and consensus), and enzymes.These methods could be applied to any number of cell lines that are usedfor production of recombinant proteins, including other myeloma celllines, such as murine NSO or rat YB2/0; epithelial lines, such as CHOand HEK 293; mesenchymal cell lines, such as fibroblast lines COS-1 orCOS-7; and neuronal cells, such as retinal cells, as well as glial andglioma cells.

Recombinant Antibody Expression in Cells Expressing Apoptosis Inhibitors

Prior work has described the effects of co-expressing Bcl-2, a naturallyoccurring apoptosis inhibitor, in recombinant CHO cells producing achimeric antibody. (See Tey et al., Biotechnol. Bioeng. 68:31-43(2000).) Although increased cell culture life was observed, antibodyproduction did not increase over equivalent cells that lacked Bcl-2expression. However, the present inventors have found that production ofrecombinant antibody from myeloma cells is significantly increased whenthe cells also express Bcl-2.

Advantageously, the myeloma cell line is stably transfected with anexpression cassette encoding the antibody or antibody fragment. Asuitable expression cassette contains one or more promoters thatcontrols expression of the antibody heavy and light chains (of singlechain in the case of an scFv) together with a selectable marker asdescribed above. A particularly useful vector is pdHL2, which contains aselectable marker gene comprising a promoter operatively linked to a DNAsequence encoding a selectable marker enzyme; a transcription unithaving a promoter operatively linked to a DNA sequence encoding theprotein of interest; an enhancer element between the selectable markergene and the transcription unit, which stimulates transcription of boththe selectable marker gene and the first transcription unit compared tothe transcription of both the selectable marker gene and the firsttranscription unit in the absence of the first enhancer.

The vector also contains a blocking element composed of a promoterplaced between the first enhancer and the selectable marker gene, whichis potentially useful for selectively attenuating the stimulation oftranscription of the selectable marker gene. V_(H) and V_(L) sequencescan be ligated into pdHL2, which is an amplifiable vector containingsequences for the human light chain constant region, the heavy chainconstant region, and an amplifiable dhfr gene, each controlled byseparate promoters. See Leung et al., Tumor Targeting 2:184, (1996) andLosman et al., Cancer 80:2660-2667, (1997). This vector can betransfected into cells by, for example, electroporation. Selection canbe performed by the addition of 0.1 μM or a suitable concentration ofmethotrexate (MTX) into the culture media. Amplification can be carriedout in a stepwise fashion with increasing concentration of MTX, up to 3μM or higher. Cells stably transfected with the expression cassette andthat constitutively express the antibody of interest can therefore beobtained and characterized using methods that are well known in the art.See also Example 4, below. After selection and cloning, theantibody-expressing cell line can then be transfected with an expressionvector that encodes an anti-apoptosis gene, such as Bcl-2. For example,the vector pZeoSV (Invitrogen, Carlsbad, Calif.) containing the Bcl-2gene fused to an SV40 promoter is transfected into the cell using asuitable method such as electroporation, and selection and geneamplification can be carried out if necessary.

Alternatively, a suitable host cell may be transfected with an apoptosisinhibitor, such as a mutant Bcl-2 gene, then adapted for growth inserum-free medium prior to further transfection, preferably inserum-free medium, with an expression vector encoding a desired proteinof interest. Antibody production using the resulting cell line can becarried out as above and compared to production in cells that do notexpress an apoptosis inhibitor.

Representative examples to illustrate the present invention are givenbelow. Example 1 describes that the incorporation of HPV-16 E6/E7 intoSp2/0 cell leads to an improved cell clone, Sp-E26, showingcharacteristics of reduced/delayed apoptosis. Example 2 describes amethod to improve host cell lines by over-expression of the HPV-16 E7element alone. Example 3 describes using Sp-E26 as a host to developtransfectants producing a recombinant Ab. Example 4 describes theenhanced production of Mab observed for an antibody-producing cell linethat co-expresses the E6/E7 element. Example 5 describes the generationand characterization of a modified Sp2/0 cell line (designated Sp-EEE)that constitutively expresses a mutant Bcl-2 (Bcl-2-EEE) possessingthree point mutations, resulting in improved longevity. Example 6describes the improved growth properties of an antibody-producing cellline that expresses Bcl-2. Example 7 describes the enhanced productionof MAb observed for the Bcl-2 expressing cell line of Example 6. Example8 describes the methods to improve a cell clone producing low-levelrecombinant protein by introduction of Bcl-2 expression in the cell.Example 9 describes the methods to improve Sp-E26 by introduction ofBcl-2 expression in the cell. Example 10 describes the use of Sp-EEE asa host to develop transfectants producing a recombinant Ab. Example 11describes the use of fed-batch reactor profiles and feeding schedules tooptimize yield. Example 12 describes the generation of a subclone ofSp-EEE capable of growth as well as transfection in serum-free media.

While preferred embodiments are illustrated herein by way of cell linestransfected with one or more genes encoding inhibitors of apoptosisknown in the art, the skilled artisan will realize that in alternativeembodiments, various substitutions, deletions or insertions may be madein the coding and/or non-coding sequence of such genes within the scopeof the claimed methods and compositions, so long as the encoded proteinexhibits the same physiological function (anti-apoptosis) as the nativeprotein. In certain embodiments, the encoded protein(s) may exhibit 80%or greater sequence identity with the native (wild-type) protein, morepreferably 85% or greater, more preferably 90% or greater, morepreferably 95% or greater, more preferably 98% or greater, morepreferably 99% or greater, most preferably 99.5% or greater sequenceidentity.

Antibodies

Various embodiments may concern antibodies and/or antibody fragmentsexpressed from the transfected cell lines of interest. The term“antibody” is used herein to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlowe and Lane, 1988, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory). Antibodies of use may also becommercially obtained from a wide variety of known sources. For example,a variety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and areavailable for use in the claimed methods and compositions. (See, forexample, U.S. Pat. Nos. 7,060,802; 7,056,509; 7,049,060; 7,045,132;7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133;7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863;6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924;6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645;6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879;6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812;6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227;6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778;6,812,206; 6,793,924; 8,783,758; 6,770,450; 6,767,711; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,652,852; 6,635,482;6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852;6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618; 6,545,130;6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404;6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408; 6,479,247;6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; 6,455,044;6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402;6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,274;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459 each incorporated herein by reference with respect to the ATCCdeposit number for the antibody-secreting hybridoma cell lines and theassociated target antigens for the antibodies or fragments thereof.)These are exemplary only and a wide variety of other antibody-secretinghybridomas are known in the art. The skilled artisan will realize thatantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, PubMed and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transformed into an adapted host cell and used for protein production,using standard techniques well known in the art.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Exemplary methods for producing antibody fragmentsare disclosed in U.S. Pat. No. 4,036,945; U.S. Pat. No. 4,331,647;Nisonoff et al., 1960, Arch. Biochem. Biophys., 89:230; Porter, 1959,Biochem. J., 73:119; Edelman et al., 1967, METHODS IN ENZYMOLOGY, page422 (Academic Press), and Coligan et al. (eds.), 1991, CURRENT PROTOCOLSIN IMMUNOLOGY, (John Wiley & Sons).

Other methods of forming antibody fragments, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments or other enzymatic, chemical or genetic techniques also maybe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association can benoncovalent, as described in Inbar et al., 1972, Proc. Nat'l. Acad. Sci.USA, 69:2659. Alternatively, the variable chains may be linked by anintermolecular disulfide bond or cross-linked by chemicals such asglutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell. The recombinanthost cells synthesize a single polypeptide chain with a linker peptidebridging the two V domains. Methods for producing sFv's are well-knownin the art. See Whitlow et al., 1991, Methods: A Companion to Methods inEnzymology 2:97; Bird et al., 1988, Science, 242:423; U.S. Pat. No.4,946,778; Pack et al., 1993, Bio/Technology, 11:1271, and Sandhu, 1992,Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.). Where an antibody-secreting hybridoma cell line ispublicly available, the CDR sequences encoding antigen-bindingspecificity may be obtained, incorporated into chimeric or humanizedantibodies, and used.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immunol., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990).

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods, as known in the art. The skilled artisanwill realize that this technique is exemplary only and any known methodfor making and screening human antibodies or antibody fragments by phagedisplay may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled to other molecules bychemical cross-linking or other known methodologies. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXenoMouse® system but may utilize any transgenic animal that has beengenetically engineered to produce human antibodies.

EXAMPLES Example 1 Generation of Apoptosis-Resistance Cell Clones byStable Expression of HPV-16 E6 and E7 Genes

Selection of Cell Clones Resistant to CHX Treatment

Sp2/0 cells were transduced with an LXSN retroviral vector containingthe expression cassette of HPV-16 E6 and E7 genes at an MOI (multiple ofinfection) of 10:1. After recovery for 24 h, the infected cells wereselected in G418 (1000 μg/ml) for 10 days. G418-resistant cells werecloned in 96-well cell culture plates by limiting dilution (0.5cells/well). Stable infectants were screened for resistance to treatmentby cycloheximide (CHX), a potent apoptosis-inducing agent. Briefly,healthy cells (viability>95%, FIGS. 1C and D) were incubated in mediumcontaining 25 μg/ml of CHX and cell morphology was examined under amicroscope. While more than 50% of parent Sp2/0 cells underwentmorphology change after two to three hours of incubation and becamefragmented (FIG. 1A), several E6/E7 transfected clones showed lessextent of morphology change, indicating resistance to apoptosis. Thebest clone, designated as Sp-E26, showed no apparent morphology changeupon four hours of treatment (FIG. 1B).

To avoid tedious visual examination, the MTT assay was used to accessthe changes in viable cell population. After the healthy cells wereincubated with or without CHX under normal culture condition for 2-3 h,MTT dye was added to the wells. After further incubation for two hours,the cells were solubilized by adding a lysis buffer contain SDS and HCl.The plates were incubated overnight at 37° C. and OD reading wasperformed at 590 nm using an ELISA plate reader. As shown in FIG. 2, theviable cell population was significantly reduced when Sp2/0 cells weretreated with CHX. By comparison, under the same treatment conditions(concentration of CHX and length of time), Sp-E26 cells tolerated betteragainst CHX treatment. With this method, a large number of clones can bescreened and selected for further analyses (FIG. 2).

Anti-Apoptosis Property of Sp-E26

CHX-induced apoptosis in Sp-E26 and the parent Sp2/0 cells was evaluatedby Annexin V staining and DNA fragmentation assay. After being incubatedin the medium containing 25 μg/ml of CHX, the cells were harvested andstained with Guava Nexin reagent (equivalent of Annexin V staining) andanalyzed in a Guava Personal Cell Analysis system (Guava Technologies,Inc.). FIG. 3 shows that while more than 30% of Sp2/0 cells becameAnnexin V positive when exposed to CHX treatment for about 1.5 h,indicating apoptosis, while Sp-E26 remained healthy, showing no increasein early apoptotic cells.

The induction of apoptosis by CHX can be revealed by analysis of theformation of intracellular oligonucleosomal DNA fragments, a hallmark ofapoptosis. The cellular DNA was extracted from CHX-treated and untreatedSp-E26 and Sp2/0 cells and DNA laddering assay was performed. In Sp2/0cells treated with CHX, extensive DNA fragmentation was detected (FIG.4). In contrast, under identical treatment conditions, the genomic DNAof Sp-E26 was still intact, showing no appearance of DNA fragmentation(FIG. 4).

Presence of HPV E6 and E7 Genes in Sp-E26 Cells

To confirm that E6 and E7 genes are stably present in the genome ofSp-E26 cells, oligonucleotide primers specific for E6 and E7 genes weredesigned and used in a PCR reaction with the genomic DNA extracted fromSp-E26 as the template, resulting in an about 700 bp DNA fragment. ThePCR product was cloned and confirmed to be E6 and E7 genes by DNAsequencing. No E6 and E7 genes were detected in the parent Sp2/0 cells.

Improved Growth Properties of Sp-E26

The growth properties of Sp-E26 were evaluated in T-flask (FIG. 5) and 3L-batch bioreactor (FIG. 6). Sp-E26 showed improved growth propertiesover the parent Sp2/0 cell in batch cultures, achieving higher maximumcell density and longer survival time.

Example 2 Generation of Apoptosis-Resistance Cell Clones by StableOver-Expression of HPV16 E7 Gene

The structure of the poly-cistronic HPV 16 E6 and E7 genes integratedinto the genome of clone Sp-E26 was analyzed by PCR using the primerpair E6-N8⁺ (ATGTTTCAGGACCCACAGGAGCGA; SEQ ID NO: 8) and E7-C8⁻(TTATGGTTTCTGAGA ACAGATGGG; SEQ ID NO: 9) and DNA sequencing. Since thesequences of primer E6-N8⁺ and E7-C8⁻ match with the coding sequence forthe N-terminal 8 amino acid residues of E6 and the complementarysequence for the C-terminal 8 codons of E7, respectively, the ampliconof full-length E6 and E7 is expected to be about 850 bp. However,amplification of the genomic DNA prepared from Sp-E26 cell with E6-N8⁺and E7-C8⁻ resulted a PCR fragment of only about 700 bp. DNA sequencingof the 700 bp PCR product revealed a deletion of a 182 poly-nucleotidefragment from the E6 gene. The defective E6 gene likely resulted fromsplicing and encodes a truncated E6 peptide with N-terminal 43 aminoacid residues. Considering the major physiological activity attributedto E6 is its ability to down-regulate p53 expression, the truncated E6protein is probably not fully functional because the level of p53expression in Sp-E26 was found to be more stable than that in Sp2/0.

Thus, to evaluate whether HPV-16 E7 gene alone is sufficient to haveanti-apoptotic effect and to improve the growth properties of Sp2/0cells, transfection of Sp2/0 cell with HPV-16 E7 is performed asfollows:

-   -   (i) The DNA sequence encoding E7 is cloned from Sp-E26 cell by        RT-PCR. Proper restriction sites are introduced to facilitate        the ligation of the gene into a mammalian expression vector,        pRc/CMV (Invitrogen). Transcription of the viral gene within the        vector, designated as E7pRc, is directed from CMV        promoter-enhancer sequences. The vector also contains a gene        conferring neomycin resistance, which is transcribed from the        SV40 promoter.    -   (ii) Sp2/0 cells are transfected with the expression vector        containing the expression cassette of HPV-16 E7 gene. Briefly, 5        μg of E7pRc is linearized by ScaI and transfected into the cell        by electroporation.    -   (iii) After recovery for 24 hours, the transfected cells are        selected in G418 (1000 μg/ml) for 10 days.    -   (iv) G418-resistant cells are then cloned in 96-well cell        culture plates by limiting dilution (0.5 cells/well). Stable        transfectants are selected and screened for resistance to        treatment by cycloheximide (CHX), a potent apoptosis-inducing        agent.    -   (v) Healthy cells (viability>95%) are incubated in medium        containing 25 μg/ml of CHX or in the absence of CHX for 3-4        hours under normal culture conditions, followed by the addition        of MTT dye into the wells. After further incubation for two        hours, the cells are solubilized by adding a lysis buffer        contain SDS and HCl. The plates are incubated overnight at        37° C. and an OD reading is performed at 590 nm using an ELISA        plate reader. Cell clones showing resistance to CHX treatment        are selected and expanded for further analyses.    -   (vi) The anti-apoptosis property of E7-transfected cells is        evaluated by Annexin V staining and DNA fragmentation assays. In        the Annexin V assay, after being incubated in the medium        containing 25 μg/ml of CHX, the cells are harvested and stained        with Guava Nexin reagent (equivalent of Annexin V staining) and        analyzed in a Guava Personal Cell Analysis system (Guava        Technologies, Inc.). In the DNA fragmentation assay, the        cellular DNA is extracted from CHX-treated and untreated        E7-transfectants and Sp2/0 cells and analyzed with agarose gel        electrophoresis.    -   (vii) Expression of the viral oncogene in E7-transfectants is        evaluated by Southern blot (genomic level), Northern blot (mRNA        level), and immunoblot (protein level) analysis. Expression of        intracellular proteins that are involved in apoptosis processes        and affected by E7 protein are examined by immunoblotting        analyses.    -   (viii) The growth properties of selected E7-transfectants are        evaluated in T-flask and in a 3 L-batch bioreactor. The        transfectants show improved growth properties, i.e. achieving        higher maximum cell density and longer survival time, over the        parent Sp2/0 cell in batch cultures are considered to be better        host cells.

Example 3 High-Level Expression of hLL2 IgG in Sp-E26

In this example, Sp-E26 is used as a host to generate cell clonesproducing hLL2, a humanized anti-CD22 Ab developed for treating patientswith NHL and autoimmune diseases. An hLL2-producing clone, 87-2-C9, waspreviously generated by using Sp2/0 cell as a host (Losman et al.,Cancer 80, 2660-2666, 1997), in which case, only one positive clone (afrequency of about 2.5×10⁻⁷) was identified after transfection, and themaximum productivity (P_(max)), defined as the concentration of theantibody in conditioned terminal culture medium in T-flask, of the onlyhLL2-producing clone, before amplification, was 1.4 mg/L. Transfectionof Sp-E26 cell with the same hLL2pdHL2 vector and by using similarprocedures as described by Losman et al. (Cancer 80, 2660-2666, 1997)resulted in more than 200 stable hLL2-producing clones, a frequency of>10⁻⁴). The P_(max) of 12 randomly selected clones was evaluated andfound to be between 13 and 170 mg/L, with a mean of 50 mg/L. Theproductivities of these clones can be further enhanced by geneamplification with MTX. This example demonstrated the advantage of usingSp-E26 over its parent Sp2/0 cell as a host for the development of cellclones producing recombinant proteins.

Example 4 Improvement of Ab-Producing Cell Lines by Stable Expression ofHPV16 E6 and E7 Genes

607-3u-8 cells were originally generated from Sp2/0 by transfection toproduce a humanized monoclonal Ab. The clone was developed by geneamplification (with MTX) and subcloning to enhance the maximum (Ab)productivity up to 150 mg/L, which decreased to about 100 mg/L followingweaning off serum supplement in the culture medium. To obtain higherantibody productivity under serum-free conditions, E6/E7 genes of HPV-16were introduced into 607-3u-8 and the effect of E6/E7 on Ab-productivitywas evaluated as follows.

607-3u-8 cells maintained in HSFM supplemented with 10% FBS and 3 μM MTXwere transduced with an LXSN retroviral vector containing the expressioncassette of HPV-16 E6 and E7 genes at an MOI of 10:1. After recovery for24 h, stably transfected cells were selected in G418 (400 μg/ml) for 10days. G418-resistant cells were subcloned in 96-well cell culture platesby limiting dilution (0.5 cells/well). A surviving clone, designated as607E1C12, was obtained for evaluation. Two subclones, designated as607-3u-8-7G7 and 607-3u-8-2D10, of 607-3u-8 without E6/E7 transfectionwere also selected. The P_(max) of these three clones were determinedand there were no significant difference (Table 1).

These results suggest that introducing E6/E7 genes into the cell doesnot alter the ability of cells producing Ab. Next, 607E1C12,607-3u-8-7G7 and 607-3u-8-2D10 were adapted to grow in serum-free mediumand the productivities of these clones were determined. All cells weregrowing well in serum-free medium. The final antibody productivity ofclone 607E1C12 was maintained at 150 mg/L, while the two clones withoutE6/E7 were substantially reduced. In addition, the productivity of607E1C12 was stable after a freeze (for cryopreservation) and thaw cycle(Table 1).

TABLE 1 The productivities of Ab-producing clones P_(max) (mg/L)^(a)Clone With serum Serum-free 607-3u-8-7G7 127 ± 16 (3)^(b)  74 ± 10 (4)607-3u-8-2D10 140 ± 4 (3)    35 ± 2 (2)  607E1C12 154 (1) 142 ± 13 (6)607E1C12 (Cryo)^(c) 145 ± 17 (5) ^(a)Determined by protein purificationof IgG from terminal culture supernatants. ^(b)The number in parenthesisindicates the sample size. ^(c)Cells had been frozen forcryopreservation.

Example 5 Generation and Characterization of a Genetically ModifiedSp2/0 Cell Line that Constitutively Expresses a Mutant Bcl-2

Evidence suggests that a mutant Bcl-2 possessing three point mutations(T69E, S70E and S87E) exhibits significantly more anti-apoptoticactivity compared to wild type or single point mutants (Deng et al.,PNAS 101: 153-158, 2004). Thus, an expression vector for this triplemutant (designated as Bcl-2-EEE) was constructed and used to transfectSp2/0 cells for increased survival and productivity, particularly inbioreactors. Clones were isolated and evaluated for Bcl-2-EEE expressionlevel, growth and apoptotic properties. The nucleic acid sequence forthe Bcl-2-EEE is depicted as SEQ ID NO. 3; the corresponding amino acidsequence for the Bcl-2-EEE protein is depicted as SEQ ID NO. 4.

A 116 bp synthetic DNA duplex was designed based on the coding sequencefor amino acid residues 64-101 of human Bcl-2. The codons for residues69, 70 and 87 were all changed to those for glutamic acid (E). Theentire sequence was extraordinarily GC rich and had numerous poly G andpoly C runs. Conservative changes were made to several codons to breakup the G and C runs and decrease the overall GC content.

Two 80-mer oligonucleotides were synthesized that, combined, span the116 bp sequence and overlap on their 3′ ends with 22 bp (See SEQ ID NOs.5 and 6). The oligonucleotides were annealed and duplex DNA wasgenerated by primer extension with Taq DNA polymerase. The duplex wasamplified using the PCR primers, Bcl-2-EEE PCR Left(TATATGGACCCGGTCGCCAGAGAAG; SEQ ID NO: 10), and Bcl-2-EEE PCR Right(TTAATCGCCGGCCTGGCGGAGGGTC; SEQ ID NO: 11).

The 126 bp amplimer was then cloned into pGemT PCR cloning vector. TheBcl-2-EEE-pGemT construct was digested with TthI and NgoMI restrictionendonucleases and the 105 bp fragment was gel isolated and ligated withhBcl-2-puc19 vector (ATCC 79804) that was digested with Tthl and NgoMIto generate hBcl-2(EEE)-puc19. The sequence of this construct wasconfirmed.

A 948 bp insert fragment was excised from hBcl-2 (EEE)-puc19 with EcoRIand ligated with pZeoSV2+ vector that was digested with EcoRI andtreated with alkaline phosphatase. The resulting construct is hBcl-2(EEE)-pZeoSV2+.

Sp2/0 cells (5.6×10⁶) were then transfected by electroporation with 60μg of hBcl-2 (EEE)-pZeoSV2+ following the standard protocol for Sp2/0cells. The cells were plated into six 96-well plates that were incubatedwithout selection for 48 hours. After two days, 800 μg/ml of zeocin wasadded to the media.

Cells from 40 wells were expanded to 24-well plates and analyzed bywestern blot with anti-hBcl-2 and anti-beta actin. All but 5 of the 40showed medium to high levels of Bcl-2-EEE expression. The results forone of four gels are shown in FIG. 7. An Sp2/0 derived hMN 14 cell line(Clone 664.B4) that was previously transfected with wild type Bcl-2 wasused as a positive control (+). As was demonstrated by Deng et al., theBcl-2-EEE migrates slightly slower than wild type Bcl-2 in SDS-PAGE.

Three strongly positive wells (#7, #25 and #87) were chosen for furtherevaluation and sub-cloning. Limiting dilution plating resulted in <20positive wells per 96-well plate, indicating a very high probability(>99%) that the cells in individual wells are in fact cloned. Initially,23 subclones from the three original wells were analyzed by GuavaExpress using anti-hBcl-2-PE (FIG. 8). The results confirmed that theoriginal wells contained mixed cell clones. Well #7 yielded clones withthe strongest signal and well #25 had those with the lowest. Clones7-12, 7-16, 87-2 and 87-10 were expanded for further analysis.Subsequently, some initially slower growing subclones were similarlyanalyzed and one clone, 87-29, gave a signal that was 20% higher thanany other clone and was expanded for further analysis.

Two high expressing SP-EEE clones (87-29 and 7-16) were compared to theuntransfected Sp2/0, Raji and Daudi cells (FIG. 9). The Sp-EEE clonesexpresses about 20-fold higher than Raji and Daudi cells, which are bothknown to express Bcl-2 at presumably normal cell levels. Sp2/0 cellswere negative. This was further verified by anti-Bcl-2 immunoblot (FIG.10). Bcl-2 was not detected with a human Bcl-2 specific antibody inSp2/0 cells even with high protein loading (50K cells) and long exposureof X-ray film. Immunoblot analysis with an anti-Bcl-2 MAb (C-2, SantaCruz Biotech.) that recognizes mouse, rat and human Bcl-2 did not detectany Bcl-2 from untransfected Sp2/0 cells, even with high protein loading(100K cells) and long exposure time (FIG. 10B). If there is any Bcl-2expressed in Sp2/0 cells, it is at a level that is more than 2 orders ofmagnitude less than the Bcl-2-EEE in clone 87-29.

Growth curves were compared for five Sp-EEE subclones and Sp2/0 cells.Three Sp-EEE subclones displayed a clear advantage over Sp2/0 cells.These three (7-12, 7-16 and 87-29) also express the highest levels ofBcl-2-EEE. As 7-12 and 7-16 are from the same original well and havenearly identical properties (Bcl-2-EEE levels and growth curves), theylikely originated from the same initial clone. The best two SP-EEEsubclones 7-16 and 87-29 were used for further evaluation.

The clones were plated in media supplemented with 10%, 1% or 0% serum(without weaning) and cell density and viability were monitored. In 10%serum 87-29 grew to a high density and had more than 4 days increasedsurvival compared to Sp2/0 cells (FIG. 11). In 1% serum, all cells grewto about 35-40% of the density achieved in 10% serum and the Bcl-2-EEEtransfectants had a similar survival advantage over Sp2/0 (FIG. 12).When transferred directly into serum free media, the Sp2/0 cells onlygrew to 600K cells/ml while 87-29 cells grew to a two-fold higherdensity (FIG. 13). In each serum concentration 87-29 cells survived 4-6days longer than Sp2/0 cells.

The methotrexate (MTX) sensitivity was determined for 87-29 (FIG. 14).The data suggests that a minimum MTX concentration of 0.04 μM issufficient for initial selection of MTX-resistant clones. Therefore, thesame selection and amplification protocols used for Sp2/0 cells can beemployed with the SP-EEE cells.

Bcl-2 is a pro-survival/anti-apoptotic protein. It has been demonstratedby several groups that a Bcl-2 deletion mutant missing the flexible loopdomain (FLD) has an enhanced ability to inhibit apoptotosis (Figueroa etal., 2001, Biotechnology and Bioengineering, 73, 211-222; Chang et al.,1997, EMBO J., 16, 968-977). More recently, it was demonstrated thatmutation of 1 to 3 S/T residues in the FLD of Bcl-2 to glutamic acid,which mimics phosphorylation, significantly enhances its anti-apoptoticability (Deng et al. 2004, PNAS, 101, 153-158). The triple mutant (T69E,S70E and S87E) provided the most significant survival enhancement. Here,a similar Bcl-2 triple mutant construct (Bcl-2-EEE), was used to stablytransfect Sp2/0 cells.

All the aforementioned experiments demonstrate that expression ofBcl-2-EEE reduces apoptosis rates in Sp2/0 cells. This effect waslargely dose dependent, in that clones with higher expression levelssurvived longer than those with lower levels. The best clone, 87-29,grows to a 15-20% higher cell density and survives an additional 4-6days compared to untransfected Sp2/0 cells.

The Bcl-2-EEE level in clone (87-29) is approximately 20-fold higherthan normal levels in Daudi or Raji cells. No Bcl-2 expression wasdetected in untransfected Sp2/0 cells. As described in Example 6,hMN-14-expressing Sp2/0 cells were transfected with a similar constructfor expression of wild type Bcl-2 and a clone with exceptional growthproperties and enhanced productivity was isolated. When this clone(664.B4) was amplified further with MTX, the Bcl-2 levels increasedsignificantly. Ultimately, the amplified (3 μM MTX) cell line wassub-cloned and the Bcl-2 level of one clone (664.B4.1C1) was two-foldhigher than 664.B4. This particular subclone has superior productivityand growth properties. The Bcl-2-EEE level in 87-29 is approximatelytwo-fold higher than the level of Bcl-2 in the amplified 664.B4.1C1.87-29 cells have a growth rate that is comparable to that of Sp2/0 cellsand can apparently continue to grow for one additional day and reach amaximal density that is 15-20% higher than Sp2/0. A similar property wasfound for the E6/E7 expressing Sp-E26 cell line. The Bcl-2-EEEexpressing 87-29 clone, which provides an additional 4-6 days survivalover the parental Sp2/0 cells, is superior to the Sp-E26 clone, whichonly survives one additional day.

The Sp-EEE cell line as represented by the 87-29 clone is useful as anapoptosis-resistant host for expressing a recombinant protein upontransfection with a suitable vector containing the gene for thatrecombinant protein. In order for this cell line to be useful it mustmaintain its Bcl-2-EEE expression and survival advantage followingtransfection and amplification and during extended culture. It isunlikely that the stably transfected Bcl-2-EEE gene will be lost duringsubsequent transfection and therefore the survival properties should notdiminish. It is possible that MTX amplification could even improve thesurvival of producing clones via increasing expression of Bcl-2proteins. Indeed, this was the case with the hMN-14 664.B4 cell line,which was transfected with wild type Bcl-2. Following amplification andsub-cloning, the Bcl-2 level increased several fold and cell survivalimproved significantly.

The final Sp/EEE clone (#87-29) has a growth rate that is comparable tothe parental Sp2/0 cells. However, the Sp/EEE 87-29 cells continue togrow for one additional day, reach a maximal density that is 15-20%greater and display an additional 4-6 days survival compared to Sp2/0.Further, the Sp/EEE cell line was considerably more tolerant to serumdeprivation compared to Sp2/0 cells.

Example 6 Improvement of Ab-Producing Cell Survival in Stationary BatchCulture by Stable Expression of a Human Bcl-2 Gene

Generation of a Bcl-2-Transfected Cell Clone

A cell clone 665.2B9 was originally generated from Sp2/0 by transfectionto produce a humanized monoclonal anti-CEA Ab (Qu et al., unpublishedresults). A vector, designated hMN14pdHL2, was used to transfect Sp2/0cells to obtain the cell clone 665.2B9. The pdHL2 vector was firstdescribed by Gillies et al., and had an amplifiable murine dhfr genethat allows subsequent selection and amplification by methotrexatetreatment (Gillies et al., J. Immunol. Methods 125:191 (1989)).Generally, the pdHL2 vector provides expression of both IgG heavy andlight chain genes that are independently controlled by twometallothionine promoters and IgH enhancers. A diagram of the hMN14pdHL2vector is shown in FIG. 16. SEQ ID NO. 1 shows the sequence of thevector; SEQ ID NO. 2 shows the 72 bp sequence defined as the enhancersequence; the promoter sequence corresponds to nt2908-2979 ofhMN14pdHL2.

Sp2/0 cells can be generally transfected by electroporation withlinearized pdHL2 vectors such as the hMN14pdHL2 vector used in thisinstance. Selection can be initiated 48 hours after transfection byincubating cells with medium containing 0.05 to 0.1 μM MTX.Amplification of inserted antibody sequences is achieved by a stepwiseincrease in MTX concentration up to 5 μM.

The clone was subjected to gene amplification with MTX increasedstepwise to 0.3 μM, at which point the maximum productivity (Pmax) ofthe antibody was increased to about 100 mg/L. To improve cell growthproperties, 665.2B9 cells were transfected with a plasmid expressionvector (FIG. 17) containing the human Bcl-2 gene by electroporation.Bcl-2 gene was excised from pB4 plasmid purchased from ATCC (pB4,catalog #79804) using EcoRI sites and inserted into MCS of mammalianexpression vector pZeoSV(+) using the same restriction enzyme. Sincezeocin resistance gene is part of the vector, transfected cells wereplaced into medium containing zeocin ranging from 50-300 μg/mL. Stableclones were selected from media containing 300 mg/ml zeocin andsubcloned in media without zeocin by plating into 96-well plates at adensity of 0.5 cell/100 uL/well. The media without zeocin was usedthereafter.

Formation of clones in wells was confirmed by visual observation under amicroscope. Cells from the wells with only 1 cluster of cells wereexpanded. Each 96-well plate produced around 30 clones, from which 14clones were randomly selected for further studies. The growthcharacteristics of these clones were evaluated by daily cell countingand viability measurements with ViaCount reagent and Guava PCA. From the14 clones evaluated in 24-well plates (FIGS. 18, 19), oneBcl-2-transfected clone showing improved growth characteristics (highercell densities and prolonged cell survival) was identified anddesignated as 665.2B9#4 (or clone #4). Comparing to the parent 665.2B9clone, clone #4 grew to a higher cell density (about 1.7-fold) andsurvived 4 to 6 days longer in T-flasks (FIGS. 20, 21), and as aconsequence of better growth, the P.sub.max of clone #4 was increased toabout 170 mg/L as determined by ELISA titration and Protein A columnpurification.

Bcl-2 Expression in 665.2B9#4

To confirm that the improved growth properties of 665.2B9#4 wereresulted from transfection of Bcl-2, intracellular level of human Bcl-2protein was measured by using Guava Express reagent and Guava PCAinstrument. Briefly, 4×10.sup.5 cells placed in 1.5 ml spin-tubes werecentrifuged for 5 minutes at 1500 rpm, washed three times with 1×PBS.Supernatants were carefully aspirated. Fixation solution (10×, 60 μL)from Santa Cruz Biotechnology (SCB), Inc. (cat. # sc-3622) was added tocell pellets for 15 min and incubated on ice. Fixation solution wasremoved with 4×1 mL PBS at 4° C., each time spinning as described.

Permeabilization buffer (0.5 mL) at −20° C. (SCB cat. # sc-3623) wasadded dropwise while vortexing, followed by 15 min incubation on ice.Cells were then spun and washed two times with 0.5 mL FCM wash buffer(SCB cat. # sc-3624). Final cell pellet was resuspended in 100 μL of FCMwash buffer and stained for Bcl-2 intracellular protein with 10 μL ofanti-Bcl-2 mouse monoclonal antibody conjugated to PE (obtained fromSCB). Incubation was performed at room temperature in dark for one hour.Two washes with 0.5 mL of FCM wash buffer followed. The final cellpellet was resuspended with 0.4 mL FCM wash buffer and the cellsanalyzed on Guava PC. Mean values of the fluorescence intensity (MFI)for each clone were compared to control staining with non-specific,isotype mouse IgGI conjugated with PE. The results summarized in Table 2confirm that clone 665.2B9#4 expresses a higher level of Bcl-2 proteincompared to the parental cell line. A zeocin-resistant clone (#13) thatshowed a similar growth profile as the parent 665.2B9 was negative forBcl-2 staining, confirming that Bcl-2 expression is necessary for theimprovement of growth.

TABLE 2 Intracellular level of Bcl-2 determined by Guava Express.Viability^(a) Mean FI Cell (%) (AU) 665.2B9 84  42 665.2B9#4 97 110Clone#13 92  14 Non-specific antibody  12 staining ^(a)Determined beforeassay to ensure healthy cells were used. ^(b)6665.2B9 cells stained withan isotype-matched mouse IgG1 antibody, PE-conjugated.

With Guava Express analysis it was found that the intensities offluorescent staining corresponding to Bcl-2 levels are rising with MTXamplification of clone 665.2B9#4, suggesting co-amplification of Bcl-2with the dhfr gene. To compare intracellular Bcl-2 levels of amplifiedcells, Western blotting analysis was performed on cell lysates of clone665.2B9#4 (Bcl-2 positive) and clone #13 (Bcl-2 negative) using ananti-human Bcl-2 antibody. Densitometric evaluation showed that Bcl-2signal of clone 665.2B9#4 growing in 1.0 μM MTX is 2× stronger than thecells in 0.6 μM MTX. A lysate of Clone #13 did not reveal the presenceof Bcl-2 protein (FIG. 22).

Example 7 Improved Ab-Production of Clone 665.2B9#4 Under Batch CultureCondition

By monitoring nutrients consumption in the cell cultures near theterminal phase, it was found that glucose and L-glutamine are the firstcomponents to be consumed. Experiments were carried out to determinewhether supplementation of these limiting nutrients would improve thefinal antibody yields. Two types of cultures were initiated: spiked fedbatch—where these limiting components were supplemented upon theirconsumption; and unfed batch—without nutrients supplementation. Testedwere Bcl-2-positive clone 665.2B9#4 growing in medium containing 0.6 and1 μM of MTX and the Bcl-2-negative clone #13 growing in 0.3 μM MTX.FIGS. 23 and 24 show the profiles of cell viability and cell density inboth culture types until they reached terminal stage. Protein yields,expressed as mg/L, are shown in Table 3. The results of this experimentsuggest that nutrient spiking improves total yield of produced antibodyabout 2-fold for all cultures.

TABLE 3 Antibody production under batch culture conditions Unfedbatch^(a) Spiked fed batch^(a) Cell/MTX (μM) (mg/L) (mg/L) 665.2B9#4/0.6117 286 665.2B9#4/1.0 156^(b) 296 Clone#13/0.3  74.1 165 ^(a)Determinedby Protein A column purification. ^(b)Average of two purifications.

Example 8 Introduction of Bcl-2 Gene into a Cell Line ProducingLow-Level of Recombinant Protein

A cell clone 482.2C4A was originally generated from Sp2/0 bytransfection to produce a bispecific Ab in the form of an IgG (anti-CEA)and two scFv (anti-DTPA) (Leung et al., J. Nuc. Med. 41: 270P, 2000;Hayes et al., Proc. Am. Asso. Cancer. Res. 43: 969, 2002), each of whichis covalently linked to the C-terminus of the IgG heavy chain. The clonewas subjected to gene amplification and had a final productivity ofabout 20 mg/L. To improve the growth property and eventually the Abproductivity, 482.2C4A cells were transfected with a plasmid expressionvector containing the human Bcl-2 gene by electroporation as describedin Example 6. The transfectants were selected in medium containing 750μg/ml of Zeocin after three weeks.

Zeocin-resistant cells were treated with 25 μg/ml of CHX for 5 hours toeliminate apoptosis-sensitive cells. Treated cells were washed twicewith fresh culture medium to remove CHX and resuspended in fresh growthmedium. After recovering for 24 h, the viable cells were cloned into96-well cell culture plates by limiting dilution (0.5 cells/well).Clones emerged in the wells in two weeks and were screened for Abproduction, resistance to CHX-induced apoptosis, as well as growthprofiles. Those clones performed better than the parent 482.2C4A in allaspects are selected and further characterized. The best performer isexpected to be more robust when growing under stress condition, resistto aging-culture-condition induced apoptosis, and have a higher maximumAb productivity (ca. 150% or better) comparing to the parent 482.2C4Acell.

Example 9 Introduction of Bcl-2 Gene into Sp-E26 for a FurtherImprovement of Cell Growth Properties

Sp-E26 cells are transfected with a plasmid expression vector containingthe human Bcl-2-EEE gene, as described in Example 5, by electroporation.The transfectants are selected in medium containing 500 μg/ml of Zeocinafter three weeks.

Zeocin-resistant cells are treated with 25 μg/ml of CHX for 5 hours toeliminate apoptosis-sensitive cells. Treated cells are washed twice withfresh culture medium to remove CHX and resuspended in fresh growthmedium. After recovering for 24 h, the viable cells are cloned into96-well cell culture plates by limiting dilution (0.5 cells/well).Clones emerge in the wells in two weeks and are screened for resistanceto CHX-induced apoptosis, as well as growth profiles. Those clonesperform better than the parent Sp-E26, as well as Sp-EEE, in all aspectsare selected and further characterized. The best performer containingHPV-16 E6/E7 and Bcl-2-EEE is expected to be more robust when growingunder stress condition and resistant to aging-culture-condition-inducedapoptosis than the parent Sp-E26 and Sp/EEE cells; therefore, it is abetter host cell for recombinant protein production.

Example 10 Improved Production of Recombinant Proteins with the Sp-EEECell Line

There are two paths that can be taken when developing a cell line withenhanced survival for production of recombinant proteins. One method,which has been accomplished quite successfully, as described in Example6, involves stable transfection of an already producing cell line with apro-survival gene, such as Bcl-2. However, this method requiresadditional transfection, selection and cloning steps, therebylengthening the cell line development process by at least two months andpossibly much more. Further, screening for the “best” clone is ratherinvolved, since a number of parameters need to be determined for eachclone, including growth/survival, Bcl-2 expression level andproductivity. Thus, only a small number of clones can be evaluated. Itis quite possible that clones with the highest productivity may not havesuperior survival and vice versa. An alternative strategy, employedhere, is to develop a parental cell line with superior growth andsurvival properties, which is subsequently transfected with theexpression vector for production of the desired protein.

Compared to Sp2/0 cells, the Sp-EEE cells continue to grow for oneadditional day, reach a maximal density that is 15-20% higher, andsurvive an additional 4-6 days in culture. The cells retain theirenhanced growth and survival properties when subsequently transfectedwith genes for the production of recombinant proteins, such as IgG,antibody fragments and fusion proteins, growth factors, such as G-CSF,GM-CFS, EPO, EGF, VEGF, cytokines, such as an interleukin family member(IL-1-IL-31), or interferon family members (such as alpha, beta or gammainterferon), oligonucleotides, peptides, hormones, enzymes, or vaccines(e.g., Hepatitis A, B or C, as well as others described above).

A DNA vector, such as pdHL2, containing one or more expression cassettesfor recombinant protein(s), such as an IgG, is used to transfect Sp-EEEcells by standard methods, such as electroporation. The transfectantsare plated in 96-well plates and clones are analyzed for proteinproduction by established techniques such as ELISA or Biacore.Productive clones are subjected to increasing concentrations of MTX inthe culture media over several months to amplify the genetic copynumber. Since the Bcl-2-EEE-expressing clones grow to about 20% highercell density and survive at least an additional 4 days as compared toclones generated in Bcl-2 negative Sp2/0 cells, the former will produceat least 20% more recombinant protein in standard flask or roller bottleculture. An even greater increase is realized in suspension, perfusionor fed-batch bioreactor cultures.

Example 11 Improved Ab-Production of Bcl-2 Transfected Clone 665.B4.1C1in Bioreactor

Both 665.2B9#4 and the parent clone 665.2B9 of Example 6 were weanedinto serum-free media. The cells were adapted to a customizedformulation of hybridoma serum-free medium (HSFM) (Immunomedics PN10070) containing 3 μM MTX by continuous subculture in T-flasks forseveral months. The adapted cells were scaled up from T-flasks to rollerbottles for banking. A master cell bank (MCB) for each cell line wascreated with 1×10⁷ viable cells in each 1-mL vial using an FBS-freecryopreservation solution composed of 45% conditioned medium (mediumthat is collected as supernatant after centrifugation of a culture inthe exponential growth phase), 10% DMSO and 45% HSFM. The MCB cell lineswere designated 665.2B9.1E4 (without Bcl-2 gene) and 665.B4.1C1 (withBcl-2 gene), respectively. The growth properties and antibody productionof these two clones were compared under batch culture conditions.

Experiments were conducted in 3-L bench-scale bioreactors using theabove cells expanded from the MCB. The 3-L bioreactor system is thescale-down model of a 2500-L cGMP bioreactor system. Therefore, theevaluation results would support the suitability of these cell lines forlarge-scale commercial manufacturing.

The same growth HSFM as that used in creating the MCB (Immunomedics PN10070) was used to maintain the cell line and prepare the inoculum.Basal HSFM, a customized formulation based on the growth HSFM withcustomized modifications (Immunomedics PN 10194), was used in the 3-Lfed-batch bioreactor process. Both media contain insulin and transferrinas the only trace proteins. Additional 0.1% Pluronic F68 wasincorporated into the formulation to protect cells from shear caused byagitation and aeration. This media also contained 3 μM MTX.

The specific characteristics of the continuous feeding solutions and thepulse feeding solutions are shown in tables 4 and 5 as follows:

TABLE 4 Continuous feeding solutions Solutions Formulation (Dissolve inWFI unless specified) Glucose and glutamine Glucose, 13.3 g/L;Glutamine, 20 mM solution (G/G) ImmuC2 solution Glucose, 13.3 g/L;Glutamine, 20 mM; PNS A, 50 ml/L; NaOH, 50 mM ImmuC5 solution Glucose,26.6 g/L; Glutamine, 40 mM; PNS A, 100 ml/L; NaOH, 100 mM

TABLE 5 Pulse feeding Solutions Formulation Solutions (Dissolve in WFIunless specified) TC Soy Plus  120 g/L Linoleic acid/cyclodextrin  1.5mg/ml HD lipid 500X β-mercaptoethanol/EDTA BME, 0.01M; EDTA, 0.1 mM.ImmuVitamin MEM Vitamin Solution (100x), as solvent; Choline Chloride,500 mg/L; Myo-inositol, 600 mg/L. TEC solution Transferrin solution (4mg/mL), as solvent; CaCl2, 125 mM; Ethanolamine-HCl 1 g/L Insulin 4mg/ml

The fed-batch experiments were conducted in 3 L Bellco spinner-flaskbioreactor systems (Bellco glasses, Vineland, N.J.) with 2 L of workingvolume. The bioreactor temperature, pH and dissolved oxygen (DO) weremonitored and controlled by single loop controllers. The reactortemperature was controlled at 37° C. by a heating blanket. The culturepH was controlled at 7.3 by the addition of CO₂ or 6% Na₂CO₃. Aerationwas performed through a cylindrical sintered sparger at 10 ml/min. DOwas controlled above 40% of air saturation by intermittent sparging ofO₂ into the medium. A constant agitation rate of 50 about 60 rpm wasused throughout the cultivation.

A frozen vial from MCB was thawed and recovered in T-flasks inapproximately 1 to 2 weeks. The cells were then expanded from T-flasksto roller bottles prior to inoculation into the bioreactors. Cells werecultured at 37° C. in a 5% CO₂ atmosphere and maintained in theexponential growth phase throughout the expansion process.

Prior to the inoculation, 1.2 liters of Basal HSFM was pump-transferredinto the bioreactor aseptically. The medium was air saturated tocalibrate the dissolved oxygen (DO) probe. A medium sample was alsotaken to calibrate the pH probe. Once pH probes and DO probes werecalibrated, both controllers were set to AUTO modes. Once the systemreached set points of pH (7.3) and temperature (37° C.), calculatedamount of inoculum from roller bottle was pump transferred into thebioreactor. The post-inoculation viable cell density (VCD) was around2×10.sup.5 vial cells/ml.

The feeding strategy is as follows. During the cultivation, concentratednutrient solutions were fed into the bioreactor to provide the cellswith necessary and non-excessive nutrients. Concentrated nutrientsolutions were delivered to the culture via continuous feeding and pulsefeeding. The continuous feeding solutions were pump transferred into thereactor continuously using peristaltic pumps (Watson-Marlow 101U/R). Thepulse feeding solutions were pulse fed once a day into the culture.

Two fed-batch feeding strategies were developed and applied to both celllines. Process #1 does not feed recombinant insulin during thecultivation. Process #2 is designed based on Process #1 with a modifiedlinoleic acid and lipid feeding schedule and an additional feeding ofinsulin. The following tables summarize the feedings of both processesfor both cell lines.

TABLE 6 Process #1 for cell line 665.2B9.1E4 Continuous feeding ExpectedViable Continuous Feeding Rate (ml/day) Cell Density Glucose and Day(cells/mL) Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 60 0 Day 3 pm 1.01~2.5E6 0 90 0 Day 4 am 2.51~3.5E6 0 90 0Day 4 pm 2.51~4.5E6 0 150 0 Day 5 am 4.51~6.5E6 0 0 90 Day 5 pm4.51~7.5E6 0 0 120 Day 6 7.51~12E6 0 0 120 Day 7 if <13E6 0 0 120if >13.1E6 0 0 150 Pulse feeding Pulse Feeding (mL) TC Soy Plus LA/CDLipid TEC Immu BME/ Day (120 g/L) (1.5 mg/ml) (500X) Solution VitaminEDTA Day 3 12.5 4 3 — — 15 Day 4 25 8 — — — — Day 5 50 12 — 4 15 15 Day6 60 8 2 8 — — Day 7 60 — 1 — — — Day 8 25

TABLE 7 Process #2 for cell line 665.2B9.1E4 Continuous feeding ExpectedViable Continuous Feeding Rate (ml/day) Cell Density Glucose and Day(cells/mL) Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 60 0 Day 3 pm 1.01~2.5E6 0 90 0 Day 4 am 2.51~3.5E6 0 90 0Day 4 pm 2.51~4.5E6 0 150 0 Day 5 am 4.51~6.5E6 0 0 90 Day 5 pm4.51~7.5E6 0 0 120 Day 6 7.51~12E6 0 0 120 Day 7 if <13E6 0 0 120if >13.1E6 0 0 150 Day 8 if <10E6 0 0 90 if 10.1~13E6 0 0 120 if >13.1E60 0 150 Pulse feeding Pulse Feeding (mL) TC Soy LA/CD TEC Immu InsulinPlus (1.5 Lipid Solu- Vita- BME/ (4 Day (120 g/L) mg/ml) (500X) tion minEDTA mg/ml) Day 3 12.5 2 3 — — 15 — Day 4 25 4 — — — — — Day 5 50 6 — 415 15 4 Day 6 60 4 — 8 — — 8 Day 7 60 4 — — — 15 8 Day 8 50 — — — — — 8

TABLE 8 Process #1 for cell line 665.B4.1C1 Continuous feeding ExpectedViable Continuous Feeding Rate (ml/day) Cell Density Glucose and Day(cells/mL) Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 90 0 Day 3 pm 1.01~2.5E6 0 120 0 Day 4 am 2.51~3.5E6 0 0 60Day 4 pm 2.51~4.5E6 0 0 90 Day 5 am 4.51~6.5E6 0 0 120 Day 5 pm4.51~7.5E6 0 0 150 Day 6 7.51~12E6 0 0 180 Day 7, 8, 9 if <15E6 0 0 180if >15.1E6 0 0 240 Day 10 if <10E6 0 0 120 if 10.1~13E6 0 0 150if >13.1E6 0 0 180 Pulse feeding Pulse Feeding (mL) TC Soy Plus LA/CDLipid TEC Immu BME/ Day (120 g/L) (1.5 mg/ml) (500X) Solution VitaminEDTA Day 3 12.5 4 3 — — 15 Day 4 25 8 — — — — Day 5 50 12 — 4 15 15 Day6 60 8 2 8 — — Day 7 60 — 1 — — 15 Day 8 60 — — — — — Day 9 60 — — — —15 Day 10 50 — — — — —

TABLE 9 Process #2 for cell line 665.B4.1C1 Continuous feeding ExpectedViable Continuous Feeding Rate (ml/day) Cell Density Glucose and Day(cells/mL) Glutamine ImmuC2 ImmuC5 Day 2  0.4~0.7E6 60 0 0 Day 3 am 1.0~1.7E6 0 90 0 Day 3 pm 1.01~2.5E6 0 120 0 Day 4 am 2.51~3.5E6 0 0 60Day 4 pm 2.51~4.5E6 0 0 90 Day 5 am 4.51~6.5E6 0 0 120 Day 5 pm4.51~7.5E6 0 0 150 Day 6 7.51~12E6 0 0 180 Day 7, 8, 9 if <15E6 0 0 180if >15.1E6 0 0 240 Day 10 if <10E6 0 0 120 if 10.1~13E6 0 0 150if >13.1E6 0 0 180 Pulse feeding Pulse Feeding (mL) TC Soy LA/CD TECImmu Insulin Plus (1.5 Lipid Solu- Vita- BME/ (4 Day (120 g/L) mg/ml)(500X) tion min EDTA mg/ml) Day 3 12.5 2 3 — — 15 — Day 4 25 4 — — — — —Day 5 50 6 — 4 15 15 4 Day 6 60 4 — 8 — — 8 Day 7 60 4 — — — 15 8 Day 860 4 — — — — 8 Day 9 60 4 — 4 15 15 8 Day 10 60 — — — —   1− 8

During the cultivation, bioreactor samples were taken periodically foroff-line analysis. The viable cell density (VCD) and the cell viabilitywere measured by microscopic counting using a hemocytometer afterstaining with 0.4% trypan blue dye. The glucose, lactate, glutamine,ammonia concentrations were measured using a Nova Bioprofile 200. Theantibody concentration was determined by HPLC using a protein A affinitychromatography column (Applied Biosystems, P/N 2-1001-00).

The specific antibody productivity was calculated by dividing thecumulative antibody produced by the time integral of the total viablecell in the culture:

${Q_{\lbrack{MAb}\rbrack} = \frac{( {{\lbrack{Mab}\rbrack_{t\; 1} \cdot V_{t\; 1}} - {\lbrack{Mab}\rbrack_{t\; 0} \cdot V_{t\; 0}}} )}{\int_{0}^{t\; 1}{{{VCD} \cdot V}\ {t}}}},$

in which ∫₀ ^(t1)VCD·Vdt is approximated by the

${Trapezium}\mspace{14mu} {Rule}\text{:}\frac{( {{{VCD}_{t\; 0} \cdot V_{t\; 0}} + {{VCD}_{t\; 1} \cdot V_{t\; 1}}} )( {{t\; 1} - {t\; 0}} )}{2}$

By Process #1, 665.2B9.1E4 cells grew to attain a maximal VCD of 1×10⁷viable cells/ml on day 6 with 86% of viability. After day 6, VCD and V %decreased quickly and the culture was harvested on day 8. Process #2helped the culture reach a higher VCD of 1.2×10⁷ viable cells/ml andsustain one more day.

As compared to 665.2B9.1E4 cells, 665.B4.1C1 cells exhibited much bettergrowth in both processes. In Process #1, its VCD reached 2×10⁷ viablecells/ml on day 7 with 97% viability. The culture also maintained thisVCD and V % for two more days before it started to decline. The culturewas harvested on day 11. In Process #2, 665.B4.1C1 cells showed asimilar growth profile as in Processes #1. More specifically, the cellsreached the highest VCD of 2.3×10⁷ viable cells/ml and the viabilitydeclined a little slower with the harvest occurring on day 11. Thisobservation was somewhat different from the 665.2B9.1E4 cell line, whichdemonstrated a growth advantage in Process #2.

The antibody yields of two cell lines in Processes #1 and #2 werecompared. The final yield of 665.2B9.1E4 cells was 0.42 g/L in Process#1 and 0.55 g/L in Process #2. For comparison, 665.B4.1C1 cellsdelivered a higher final yield of 1.5 g/L in both processes.

The daily specific antibody productivities (per cell basis) werecalculated and the 665.2B9.1E4 cells had an average daily Q_([MAb]) ofapproximately 15 pg/cell/day throughout the course of cultivation forboth processes. The additional day of growth at the highest VCD inProcess #2 resulted in a higher final antibody concentration.

The 665.B4.1C1 cells showed a similar daily specific antibodyproductivity profile in both processes with Process #1 yielding slightlyhigher productivity. The daily Q_([MAb]) were maintained between about20 to 25 pg/cell/day until day 9. Thereafter the productivity declined.

Compared with the 665.2B9.1E4 cell line, the 665.B4.1C1 cell lineexhibited a higher specific antibody productivity of about 25pg/cell/day as compared to 15 pg/cell/day. Combining with its bettergrowth, the 665.B4.1C1 cell line tripled the final antibody yield to 1.5g/L as compared to 0.55 g/L achieved by the 665.2B9.1E4 cell line. Theseresults demonstrate the benefit of incorporating Bcl-2 gene into thehost cell line to enhance its growth and antibody yield in serum-freemedia in a bioreactor modeled for large-scale commercial preparation ofa recombinant protein, in this case an antibody for clinical use.

Example 12 Development of Sp/ESF Serum Free Pre-Adapted Cell Line

A standard protocol for cell transformation and protein production issummarized as follows. Sp2/0 cells, or Sp2/0 derived lines aremaintained in 10% FBS and transfected by electroporation with anexpression vector containing the gene of interest. While maintaining thetransfectant cell lines in media supplemented with 10% FBS, attempts aremade to amplify the expression by step-wise increases in methotrexate(MTX) in the culture media. This amplification process, which only seemsto work on occasion and typically with cell lines having lower initialproductivity, usually takes 4-8 months. Once MTX amplification iscompleted, the clones are gradually adapted for growth in serum-freemedia over a period of 3-6 months, which typically results in a loss ofproductivity of up to 50%.

Since the Sp/EEE cell line showed enhanced growth and survivalproperties as well as superior tolerance to serum deprivation, it wasdecided to explore the feasibility of developing an Sp/EEE cell linethat is pre-adapted to growth in serum-free media and use this line fortransfection, cloning and amplification. The following describes thedevelopment of the Sp/ESF (Sp/EEE serum-free) cell line. Feasibility forproduction of cloned proteins, such as antibodies or fragments, wasdemonstrated by transfection with the C-AD2-Fab-h679-pdHL2 expressionvector.

Adaptation to Growth in Serum Free Media and Subcloning

Sp/EEE cells were adapted to growth in serum-free media (SFM) over a2-month period. In an attempt to determine if transfection is feasiblein SFM without FBS, serum free-adapted Sp/EEE cells were plated in alimiting dilution to determine if they would recover from low density.Cells were plated at a concentration of 5 cells/well in the first row ofa 96-well plate and diluted 2-fold down the plate. Seven clones totalresulted from the limiting dilution. These results demonstrate that thecells can survive under the conditions necessary for transfection.Growth curve experiments were performed using four of the sevensubclones to select the clone with the most favorable growth properties.The four clones (#1, 3, 4, and 5), as well as the parental clone, weresplit to a density of 3×10⁵ cells/ml in a T25 flask in 6 ml of culturemedia. The cell viability (FIG. 25) and density (FIG. 26) were monitoreduntil zero viability was reached.

Of the subclones, #3 survived 24 hours longer than any other subclone orthe parental cell line. In addition, subclones #3 and #1 reached highermaximal cell density (3.2 to 3.3 million/mL, FIG. 26) than the otherclones. This suggests that subclone #3 may be better adapted to undergosuccessful transfection. Therefore, subclone #3 was given a newdesignation of Sp/ESF and used for subsequent transfections.

Transfection of Sp/ESF cells with h679-AD2

Based on the above data Sp/ESF cells (subclone #3) were transfected byelectroporation with 30 μg of h679-AD2-pdHL2 following standard protocolfor Sp2/0 cells. After 48 hours cells were selected with 0.1 μM MTX. Asa control Sp/EEE cells in 10% FBS were also transfected withh679-AD2-pdHL2 by electroporation under the same conditions. After 10days plates were ready for screening via ELISA using BSA-IMP-260 coatedplates. For both transfections approximately 130 of 400 wells containedpositive clones. Positive Sp/ESF cells from wells with the 40 highest ODreadings were transferred to 24-well plates and the MTX was increased to0.2 μM MTX. After the cells in the 24-well plates reached terminal,further screening by BIACORE analysis using an HSG sensorchip wasperformed. Four of the screened clones had a productivity of >50 mg/L.The highest producing clone (h679-AD2-SF #T6) had an initialproductivity of 82 mg/L. These initial productivity results were verysimilar to those obtained from a previous transfection of this constructusing Sp/EEE cells in 10% FBS.

Amplification

The h679-AD2-SF# T6 clone was selected for MTX amplification. After 2weeks the MTX concentration was increased from 0.2 μM to 0.4 μM. Afteronly 2 MTX increases, some amplification in productivity can already beobserved.

TABLE 10 MTX Concentration Productivity 0.1 μM MTX  82 mg/L 0.2 μM MTX 93 mg/L 0.4 μM MTX 103 mg/L

CONCLUSIONS

The data presented above for Sp/ESF indicate that transfection, cloningby limiting dilution and MTX amplification can all be accomplished underserum-free conditions in less than a month. This was demonstrated withthe transfection of the h679-Fab-AD2-pdHL2 expression vector, resultingin the initial very high production of 82 mg/L, which could be amplifiedto 103 mg/L in two weeks. Further amplification is expected with alonger time of MTX exposure. The initial productivity of the best clone(T6) of 82 mg/L surpasses the initial productivity of the besth679-AD2-pdHL2 clone (5D8) from the original transfection of the parentSp/EEE cell line carried out in 10% FBS, which was around 50 mg/L.Sp/ESF cells have also been transfected with EPO-DDD2-pdHL2 forproduction of erythropoietin.

As shown in Table 11, which compares the key parameters of Sp/ESF withthose of the existing PER.C6 cell line (Jones et al in Biotechnol. Prog.2003, 19: 163-168), Sp/ESP excels PER.C6 in many categories.

TABLE 11 Sp/ESP PER.C6 Parental Cell line Mouse myeloma Human embryonicretina + E1 Anti-apoptotic gene Bcl-2-EEE None Transfection MethodElectroporation Lipofectamine Efficiency 130/400 ? Growth SuspensionAdherent Medium SFM 10% FBS Screening Growth Suspension Adherent MediumSFM 10% FBS Selected clones Growth Suspension Suspension Medium SFM SFMAdaption time None 4 weeks Doubling time ~12 h 30-33 h Cell cultureVessel T-25 Roller bottle Medium SFM SFM Maximal density 3.3 × 10⁶/mL 5× 10⁶/mL Productivity 103 mg/L of Fab* 300-500 mg/L of IgG *Equivalentto 300 mg/L of IgG

1. A method of producing a protein comprising: a) transfecting amammalian cell line with a first nucleic acid encoding a mutant Bcl-2protein comprising T69E, S70E and S87E amino acid substitutions, toproduce a Bcl-EEE host cell; b) adapting the Bcl-EEE host cell to growin serum-free medium to produce a pre-adapted host cell; c) transfectingthe pre-adapted host cell in serum-free medium with a second nucleicacid encoding a recombinant protein to produce a recombinant host cell;d) culturing the recombinant host cell in culture medium to produce therecombinant protein.
 2. The method of claim 1, further comprisingculturing the Bcl-EEE host cell in the presence of methotrexate toamplify the first nucleic acid.
 3. The method of claim 1, furthercomprising culturing the recombinant host cell in the presence ofmethotrexate to amplify the first and second nucleic acids.
 4. Themethod of claim 1, further comprising storing the pre-adapted Bcl-EEEhost cell frozen in serum-free medium before it is transfected with thesecond nucleic acid.
 5. The method of claim 1, wherein the culturemedium is serum-free culture medium.
 6. The method of claim 1, whereinthe recombinant protein is an antibody, an antibody fragment, a growthfactor, a protein or peptide hormone, an interleukin, an interferon oran enzyme.
 7. The method of claim 6, wherein the protein is selectedfrom the group consisting of EPO, G-CSF, GM-CSF, EGF, VEGF,thrombopoietin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, interferon-alpha, interferon-beta and interferon-gamma. 8.The method of claim 1, wherein said culture medium further comprises atleast one caspase inhibitor.
 9. The method of claim 8, wherein saidcaspase inhibitor is selected from the group consisting of caspase-1,caspase-3, caspase-9, caspase-12 and pan-caspase inhibitors.
 10. Themethod of claim 9, wherein said caspase inhibitor is selected from thegroup consisting of Z-VAD-fmk, Ac-DEVD-cho (SEQ ID NO: 7), Aven andXIAP.
 11. The method of claim 1, wherein said culture medium furthercomprises at least one growth factor, cytokine or hormone.
 12. Themethod of claim 11, wherein the growth factor, cytokine or hormone isselected from the group consisting of erythropoietin, thrombopoietin,IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, IL-13, IL-15,prolactin, growth hormone, G-CSF and GM-CSF.
 13. The method of claim 1,wherein the cell line is selected from the group consisting of Sp2/0, anSp2/0 derivative, NS0, YB2/0, CHO, HEK 293, COS-1, COS-7, HepG2, BHK21,P3X3Ag8.653 and BSC-1.
 14. The method of claim 1, wherein therecombinant host cell can be cultured in serum-free medium withoutfurther adaptation to serum-free growth.